Previously, we reported that deficits in self-renewal and bone formation capacity in bone marrow-derived mesenchymal stem cells (BM-MSCs) from elderly could be completely restored by culture on extracellular matrix (ECM) synthesized by BM stromal cells from young donors (young-ECM). However, this restorative effect was substantially diminished when cells from young or old donors were cultured on ECM synthesized by BM stromal cells from old donors (old-ECM). The goal of this renewal application is to dissect out the effective component(s) present in young-ECM that play(s) a critical role in the retention of BM-MSC properties and rejuvenation of BM-MSCs from elderly donors (old MSCs). To identify differences in protein composition between the two young- and old-ECMs, we performed proteomic analyses and discovered that CCN1/Cyr61 was the only protein present in young-ECM that was not in old-ECM. Although CCN1/Cyr61 has been shown to regulate osteoblast differentiation when added exogenously to cell cultures, it is not clear whether it can also function as a structural protein (incorporated during BM-ECM synthesis) and promote the retention of MSC properties. In the renewal application, we hypothesize that the presence of CCN1/Cyr61 in young-ECM is essential for the retention of MSC properties and rejuvenation of elderly BM-MSCs. We are well-positioned to test this hypothesis since we have established a unique 3D native BM-ECM culture system. To test our hypothesis, we will evaluate the role of CCN1/Cyr61 in controlling the behavior of MSCs by decreasing its incorporation into the matrix during synthesis of young-ECM (Specific Aim 1) and increasing its incorporation into the matrix during synthesis of old-ECM (Specific Aim 2). To determine whether CCN1/Cyr61 alone is sufficient to rejuvenate elderly MSCs, recombinant human CCN1/Cyr61 will be added to cell culture media, physisorbed onto tissue culture plastic (TCP) surfaces, or incorporated into self-assembled monolayers (SAMs) (Specific Aim 3). The proposed studies are innovative in that we will for the first time: a) demonstrate the function of CCN1/Cyr61, incorporated into BM-ECM (the BM-MSC microenvironment) as a structural protein during synthesis, in controlling the fate of authentic BM-MSCs, and b) construct a defined ECM, using recombinant CCN1/Cyr61, that mimics the ability of native BM-ECM to produce large-scale expansion of high- quality MSCs and rejuvenation of elderly MSCs. If successful, the results of the proposed studies will be significant because they will overcome challenges related to our established 3D native BM-ECM and be more suitable for clinical applications (e.g: variations in ECM prepared from different donors; safety concerns related to potential contamination with undetected pathogens; and undefined ECM components that may produce variable/unpredictable clinical outcomes). According to the 2015 National Survey of Veteran Enrollees' Health and Reliance on VA, 47% of the US veteran population is 65 years or older, creating a major challenge for treating a large number patients with age-related diseases. The information gained from the proposed studies will be important for developing a defined culture system that can be used to quickly expand large numbers of high-quality autologous MSCs for personal stem cell banks, allowing serial administration of ?rejuvenated? autologous MSCs that not only replace aged cells but also gradually reverse the aged microenvironment and delay the progression of age-related diseases or the aging process itself.